Ifnar2 mutants, their production and use

ABSTRACT

The present invention relates to mutant polypeptides of the beta chain of the type I IFN receptor (IFNAR2 mutant) with enhanced affinity for IFNβ as compared to the wild type protein for prolonging the effect of IFNβ in vivo.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/500,521, filed Jun. 30, 2004, which is the U.S. National Stage ofPCT/IL02/01059, filed Dec. 31, 2002, which claims priority to IsraelPatent Application 147414, filed Dec. 31, 2001, each of which isincorporated herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to mutant polypeptides of the beta chainof the type I IFN receptor (MIFNAR2) with enhanced affinity forinterferon-β as compared to the wild type protein for prolonging theeffect of IFNβ in vivo.

BACKGROUND OF THE INVENTION

Interferons are classified either as the leukocyte and fibroblastderived Type I interferons, or as the mitogen induced or “immune” TypeII interferons (Pestka et al, 1987). Through analysis of sequenceidentities and common biological activities, type I interferons includeinterferon alpha (IFNα), interferon beta (IFNβ) and interferon omega(IFNω), while type II interferon includes interferon gamma (IFNγ).

The IFNα, IFNβ and IFNω genes are clustered on the short arm 25 ofchromosome 9 (Lengyl, 1982). There are at least 25 non-allelic IFNαgenes, 6 non-allelic IFNω genes and a single IFNβ gene. All are believedto have evolved from a single common ancestral gene. Within species,IFNα genes share at least 80% sequence identity with each other. TheIFNβ gene shares approximately 50% sequence identity with IFNα; and theIFNω gene shares 70% homology with IFNα (Weissman et al, 1986; Dron etal, 1992). IFNα has a molecular weight range of 17-23 kDa (165-166 aminoacids), IFNβ, about 23 kDa (166 amino acids) and IFNω, about 24 kDa (172amino acids).

Type I interferons are pleiotropic cytokines having activity such ashost defense against viral and parasitic infections, anti-cancerproperties and as immune modulators (Baron et al, 1994; Baron et al,1991). Type I interferon physiological responses includeanti-proliferative activity on normal and transformed cells, stimulationof cytotoxic activity in lymphocytes, natural killer cells andphagocytic cells, modulation of cellular differentiation, stimulation ofexpression of class I MHC antigens, inhibition of class II MHC, andmodulation of a variety of cell surface receptors. Under normalphysiological conditions, IFNα and IFNβ (IFNα/β) are secretedconstitutively by most human cells at low levels with expression beingup-regulated by addition of a variety of inducers, comprising infectiousagents (viruses, bacteria, mycoplasma and protozoa), dsRNA, andcytokines (M-CSF, IL-1α, IL-2, TNFα). The actions of Type I interferonin vivo can be monitored using the surrogate markers, neopterin, 2′, 5′oligoadenylate synthetase, and β32 microglobulin (Alam et al, 1997;Fierlbeck et al, 1996; Salmon et al, 1996).

Type I interferons (IFNα/β/ω) act through a cell surface receptorcomplex to induce specific biologic effects, such as anti-viral,anti-tumor, and immune modulators. The type I IFN receptor (IFNAR) is ahetero-multimeric receptor complex composed of at least two differentpolypeptide chains (Colamonici et al, 1992; Colamonici et al, 1993;Platanias et al, 1993). The genes coding for these chains are found onchromosome 21, and their proteins are expressed on the surface of mostcells (Tan et al, 1973). The receptor chains were originally designatedalpha and beta and have been renamed IFNAR1 for the alpha subunit andIFNAR2 for the beta subunit. In most cells, IFNAR1 (alpha chain, Uzesubunit) (Uze et al, 1990) has a molecular weight of 100-130 kDa, whileIFNAR2 (beta chain, β_(L), IFNα/βR) has a molecular weight of 100 kDa.In certain cell types (monocytic cell lines and normal bone marrowcells) an alternate receptor complex has been identified, where theIFNAR2 subunit (β_(S)) is expressed as a truncated receptor with amolecular weight of 51 kDa. The IFNAR1 and IFNAR2 β_(S) and β_(L)subunits have been cloned (Novick et al, 1994; Domanski et al, 1995).The IFNAR2 β_(S) and β_(L) subunits have identical extracellular andtransmembrane domains; however, in the cytoplasmic domain they onlyshare identity in the first 15 amino acids. The IFNAR2 subunit alone isable to bind IFNα/β, while the IFNAR1 subunit is unable to bind IFNα/β.When the human IFNAR1 receptor subunit alone was transfected into murineL-929 fibroblasts, no human IFNαs except IFNα8/IFNαB were able to bindto the cells (Uze et al, 1990). The human IFNAR2 subunit, transfectedinto L cells in the absence of the human IFNAR1 subunit, bind humanIFNα, binding with a Kd of approximately 0.45 nM. When human IFNAR2subunits were transfected in the presence of the human IFNAR1 subunit,high affinity binding could be shown with a Kd of 0.026-0.114 nM (Novicket al, 1994; Domanski et al, 1995). It is estimated that from 500-20,000high affinity and 2,000-100,000 low affinity IFN binding sites exist onmost cells. Although the IFNAR1/2 complex (α/β_(S) or α/β_(L)) subunitsbind IFNα with high affinity, only the α/β_(L) pair appears to be afunctional signaling receptor.

Transfection of the IFNAR1 and the IFNAR2 β_(L) subunits into mouseL-929 cells, followed by incubation with IFNα2, induces an anti-viralstate, initiates intracellular protein phosphorylation, and causes theactivation of intracellular kinases (Jak1 and Tyk2) and transcriptionfactors (STAT 1, 2, and 3) (Novick et al, 1994; Domanski et al, 1995).In a corresponding experiment, transfection of the IFNAR2 βs subunit wasunable to initiate a similar response. Thus, the IFNAR2 β_(L) subunit isrequired for functional activity (anti-viral response) with maximalinduction occurring in association with the IFNAR1 subunit.

In addition to membrane bound cell surface IFNAR forms, a soluble IFNARhas been identified in both human urine and serum (Novick et al, 1994;Novick et al, 1995; Novick et al, 1992; Lutfalla et al, 1995). Thesoluble IFNAR isolated from serum has an apparent molecular weight of 55kDa on SDS-PAGE, while the soluble IFNAR from urine has an apparentmolecular weight of 40-45 kDa (p40). Transcripts for the soluble p40IFNAR2 are present at the mRNA level and encompass almost the entireextracellular domain of the IFNAR2 subunit with two additional aminoacids at the carboxy terminal end. There are five potentialglycosylation sites on the soluble IFNAR2 receptor. The soluble p40IFNAR2 has been shown to bind IFNα2 and IFNβ and to inhibit in vitro theanti-viral activity of a mixture of IFNα species (“leukocyte IFN”) andindividual Type I IFNs (Novick et al, 1995). A recombinant IFNAR2subunit Ig fusion protein was shown to inhibit the binding of a varietyof Type I IFN species (IFNαA, IFNαB, IFNαD, IFNβ, IFNα Con1 and IFNω) toDaudi cells and α/β_(S) subunit double transfected COS cells.

Type I IFN signaling pathways have been identified (Platanias et al,1996; Yan et al., 1996; Qureshi et al., 1996; Duncan et al., 1996; Sharfet al, 1995; Yang et al, 1996). Initial events leading to signaling arethought to occur by the binding of IFNα/β/ω to the IFNAR2 subunit,followed by the IFNAR1 subunit associating to form an IFNAR1/2 complex(Platanias et al., 1994). The binding of IFNα/β/ω to the IFNAR1/2complex results in the activation of two Janus kinases (Jak1 and Tyk2),which are believed to phosphorylate specific tyrosines on the IFNAR1 andIFNAR2 subunits. Once these subunits are phosphorylated, STAT molecules(STAT 1, 2 and 3) are phosphorylated, which results in dimerization ofSTAT transcription complexes followed by nuclear localization of thetranscription complex and the activation of specific IFN induciblegenes.

A randomized, double-blinded, placebo-controlled, two-year multicenterstudy demonstrated that natural human fibroblast interferon (interferonbeta) administered intrathecally (IT) is effective in reducing theexacerbations of exacerbating-remitting multiple sclerosis (MS). Themean reduction in exacerbation rate of 34 patients with MS who receivedinterferon beta administered IT was significantly greater during thestudy than that of 35 control patients who received placebo (Jacobs etal. 1987).

The pharmacokinetics and pharmacodynamics of Type I IFNs have beenassessed in humans (Alan et al, 1997; Fierlbeck et al, 1996; Salmon etal, 1996). The clearance of IFNβ is fairly rapid with thebioavailability of IFNβ lower than expected for most cytokines. Althoughthe pharmacodynamics of IFNβ has been assessed in humans, no clearcorrelation has been established between the bioavailability of IFNβ andclinical efficacy. In normal healthy human volunteers, administration ofa single intravenous (iv) bolus dose (6 MIU) of recombinant CHO derivedIFNβ resulted in a rapid distribution phase of 5 minutes and a terminalhalf-life of about 5 hours (Alam et al, 1997). Following subcutaneous(sc) or intramuscular (im) administration of IFNβ, serum levels are flatwith only about 15% of the dose systemically available. Thepharmacodynamics of IFNβ following iv, im or sc administration (asmeasured by changes in 2′5/-oligoadenylate synthetase (2′, 5′-AS)activity in PBMCs) were elevated within the first 24 hours and slowlydecreased to baseline levels over the next 4 days. The magnitude andduration of the biologic effect was the same regardless of the route ofadministration.

A multiple dose pharmacodynamic study of IFNβ has been conducted inhuman melanoma patients (Fierlbeck et al, 1996) with IFNβ beingadministrated by sc route, three times per week at 3 MIU/dose over asix-month period. The pharmacodynamic markers, 2′, 5′-AS synthetase,β₂-microglobulin, neopterin, and NK cell activation peaked by the secondinjection (day 4) and dropped off by 28 days, remaining only slightlyelevated out to six months.

Purification and refolding of the extracellular part of human IFNAR2(IFNAR2-EC) expressed in Escherichia coli and its characterization withrespect to its interaction with interferon alpha2 (IFNα2) has beenreported (Piehler and Schreiber 199A). The 25 kDa, non-glycosylatedIFNAR2-EC was shown to be a stable, fully active protein, which inhibitsantiviral activity of IFNα2. The stoichiometry of binding IFNα2 is 1:1,as determined by gel filtration, chemical cross-linking and solid-phasedetection. The affinity of this interaction was found to be about 3 nM(Piehler and Schreiber 2001). The rate of complex formation isrelatively high compared to other cytokine-receptor interactions. Thesalt dependence of the association kinetics suggests a limited butsignificant contribution of electrostatic forces towards the rate ofcomplex formation. The dissociation constant increases with decreasingpH according to the protonation of a base with a pKa of 6.7. Theaffinity of IFNβ to IFNAR2 is about two-fold higher than that of IFNα2to IFNAR2 (Piehler and Schreiber 1999B).

Single mutations in the binding site of IFNAR2 allowed mapping ofdifferences in binding of IFα2 and IFNβ (Piehler and Schreiber 1999B).For example, a mutation H78A was found to stabilize the complex withIFNβ nearly by two fold, while destabilized the complex with IFα2 morethan two fold. A mutation N100A was found to hardly affect the rates forbinding IFα2, whereas it decreased the dissociation rate constant forIFNβ by almost four fold.

EP1037658 discloses that the in vivo effect of Type I interferon (IFN)can be prolonged by administering the interferon in the form of acomplex with an IFN binding chain of the human interferon alpha/betareceptor (IFNAR) i.e. IFNAR behaves as a carrier protein for IFN. Such acomplex also improves the stability of the IFN and enhances the potencyof the IFN. The complex may be a non-covalent complex or one in whichthe IFN and the IFNAR are bound by a covalent bond or a peptide.EP1037658 also discloses that storing IFN in the form of such a compleximproves the storage life of the IFN and permits storage under milderconditions than would otherwise be possible.

There exists a need for an IFNAR2 with improved affinity towards IFNβ,but not to IFNα2, making IFNAR2 a better and specific carrier for IFNβ.

SUMMARY OF THE INVENTION

The invention provides an IFNAR2 mutant polypeptide (MIFNAR2) mutated atamino acid residues histidine 78 and asparagine 100, having higheraffinity for interferon-β (IFNβ) than the wild type polypeptide, or ananalog, functional derivative, fusion protein or salt thereof. Themutations are substitutions of amino acids, preferable conservativeamino acids, more preferable alanine, aspartic acid or histidine. TheIFNAR2 mutant has about 25, preferably 50 and more preferably 100-foldhigher affinity than the wild type protein and a preferred Kd of about30 pM.

More particularly the invention provides an IFNAR2 mutant polypeptidefragment comprising the extracellular domain.

In addition the invention provides a DNA encoding the IFNAR2 mutantpolypeptide of the invention, a vector comprising said DNA, host cellscomprising said vector and methods for producing a polypeptide mutant ofthe invention by cultivating said host cells and isolating the producedpolypeptide mutant.

In another aspect the invention provides the use of an IFNAR2 mutantpolypeptide for the manufacture of a medicament for modulating theeffects of IFN, preferably IFNβ, in vivo.

The invention also provides a pharmaceutical composition comprising atherapeutically effective amount of an IFNAR2 mutant or itsextracellular domain fragment, to be administrated alone orco-administrated with IFN, more preferably IFNβ, separately orcovalently bound. More specifically the invention providespharmaceutical compositions for augmenting the anti-viral, anti-cancerand immune modulating properties of IFNβ and for treatment of autoimmunediseases, such as multiple sclerosis, rheumatoid arthritis, myastheniagravis, diabetes, ulcerative colitis and lupus.

Furthermore, the invention provides methods of treatment of autoimmunediseases, viral disease and cancer, comprising administration of anIFNAR2 mutant polypeptide of the invention.

In addition the invention provides the use of IFNAR2 mutant polypeptide,preferably co-administered with an IFN antagonist, for inhibition of IFNactivity in a disease which is aggravated or caused by IFN.

The invention also provides the use of the IFNAR2 mutant polypeptide ofthe invention in a formulation to prevent IFN oligomerization.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a simulation of the concentrations of bound and free IFNβusing a constant concentration of IFN (50 pM) and increasingconcentrations of wild type IFNAR2 EC (left) and mutant IFNAR2 EC(right), with a Kd of 3 nM and 50 pM, respectively, calculated inaccordance with the law of mass action.

FIG. 2 shows the amino acid sequence of the extracellular domain of theIFNAR2 protein (not including the leader sequence) (SEQ ID NO: 1) andthe mutated amino acid residues (marked with an asterisk).

FIG. 3 shows the binding of IFNβ and IFNα2 to the IFNAR2 EC H78A/N100Amutant. Association and disassociation of IFNβ and IFNα2 to the wildtype IFNAR2 EC (upper panel), to the IFNAR2 EC H78A/N100A mutant (middlepanel) and the binding of the wild type and mutant IFNAR2 EC H78A/N100Amutant to IFNβ (lower panel) was measured using reflectometricinterference spectroscopy (RifS), with IFNAR2 immobilized to the surface(described in Piehler and Schreiber 2001). Y-axis=signal (nanometer) andthe X-axis=time (seconds).

FIG. 4 shows occlusion of IFNβ by IFNAR2 wild type and mutants. Aconstant amount of IFNβ (10 pM) was mixed with different concentrationsof IFNAR2 (R2) wild type and mutants (single mutants R2N100A and R2H78A,double mutants R2 H78A/N100A, R2 H78A/N100H and R2 H78A/N100D), and theresidual antiviral activity at equilibrium was determined in WISH cells.In the upper box, a plot of the antiviral activity of IFNβ as a functionof its concentration in the absence of IFNAR2 is shown (Y-axis 32survival index). This plot is used as a standard to determine how muchof the IFNβ is free (active) in the anti-viral assay.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a mutant of the beta chain of the type I IFNreceptor (IFNAR2), mutated at amino acid residues H78 and N 100 (see DNAsequence of wild type IFNAR2 in FIG. 2, SEQ ID N: 1) having increasedaffinity to IFNβ, but not to IFNα2 (MIFNAR2). The invention relates alsoto a drug carrier system to enhance activity of IFN comprising theextracellular domain (EC) of MIFNAR2. The invention relates to MIFNAR2,or an analog, functional derivative, fusion protein, fragment thereof orsalts thereof.

Carriers are usually administered to prolong the intra-vascularretention time of proteins having molecular weight below 50,000 daltons(e.g., interferon). Particularly beneficial are such carriers that bindnon-covalently and permit constant release of the drug. Using such acarrier is desirable in order to have at any time some portion of thefree drug available for curative activity (about 20%) and some amount ofdrug bound to the carrier and protected (about 80%).

FIG. 1 (left panel) depicts a simulation of the concentration of boundand free IFNβ in the presence of different concentrations of IFNAR2based on the law of mass action and on a Kd of 3 nM (tested byreflectometric interference spectroscopy [RifS]). This simulation showsthat in order to achieve 20% of free IFNβ (10 pM, which equals about 100Units), and 80% bound, a very high concentration of IFNAR2 protein suchas 12.5 nM (which is equivalent to 300 μg/Kg of non-glycosylated IFNAR2)is needed.

Thus, using an IFNAR2 mutant with 50 fold and higher affinities to IFNβas a carrier (see simulation FIG. 1, right panel), would be advantageoussince with such a mutant theoretically only about 0.24 nM will berequired to get 20% IFNβ free (which is equivalent to 6 μg/Kg). A mutantof the IFNAR2 with increased affinity to IFN (MIFNAR2) was generated. Toget MIFNAR2 EC, the wild type IFNAR2 EC (FIG. 1, SEQ ID NO: 1) wasmutated at two amino acid residues, residue 78 histidine and residue 100asparagine (see FIGS. 2, 3 and 4, SEQ ID NOs: 2, 3, and 4). This mutantIFNAR2 EC proteins turned out to be a better carrier specifically forIFNβ, i.e., has improved affinity for IFNβ while its affinity towardsIFNα2 remains unchanged. The affinity of the mutants for IFNβ was foundto be 26, 40 and above 50 fold higher than that of the wild type (Table4). The results obtained show that despite the increased affinity ofthis mutated soluble receptor (Kd of the H78A/N100A IFNAR2 mutant ˜30 pMversus Kd of WT protein=3 nM), enough IFNβ remains unbound andtherapeutically active, as evidenced by the anti-viral protectiveactivity of VSV challenged WISH cells (FIG. 4). The results show alsothat the levels of IFN occlusion (bound IFN at equilibrium conditions)obtainable with wild type IFNAR2 EC could be accomplished using lowerconcentrations of IFNAR EC mutants. The best results are obtained withmutants modified in both residues, particularly when both amino acidsare mutated to alanine, H78A/N100A IFNAR2, e.g., in order to get 80% ofIFNβ bound (8 pM occluded and 2 pM free IFNβ) about 30 fold lessH78A/N100A IFNAR mutant is required over the wild type IFNAR2 protein.

These results show that the double mutated IFNAR2 occludes moreeffectively IFNβ and administration of considerably lower amounts isrequired to fulfill its carrier activity towards IFNβ.

The advantages of using MIFNAR2 EC are that (I) it is possible toadminister lower quantities (thus technically feasible) of the receptoras a carrier (II) because of the stabilizing activity of the mutant itis possible to reduce the amount of IFNβ administrated, and consequentlyto reduce some of the unwanted side effects of interferon treatment(III) the increase in the activity by the mutant is specific to IFNβ,and (IV) that in some inflammatory disorders, where it may be requiredto lower the IFN concentrations, it is possible under certain conditionsto use this mutant as an effective antagonist specifically towards IFNβ,but not IFNα2.

MIFNAR2-EC may be administered alone to stabilize and enhance theactivity of endogenous IFNβ. This is particularly useful for thetreatment of patients having a disease or condition which naturallycauses the elevation of native IFN, so that the IFN will already becirculating in the body for its intended natural effect of fighting suchdisease or condition. MIFNAR2-EC will act specifically on endogenousIFNβ, but less towards IFNα2. Alternatively, MIFNAR2-EC may beco-administrated together with IFN, preferably IFNβ or may beadministrated covalently bound to IFNβ, i.e., as a complex, to modulatethe activity of IFNβ. Preferably, MIFNAR2 and IFNβ used to generate thecomplex are recombinant molecules.

The technology required to produce the fusion protein of the mutant ECIFNAR2 and IFN is similar to the technology described for wild typeIFNAR/IFN complex production which is described in detail in WO9932141,wherein the IFNAR2 mutated at H78 and N100 (MIFNAR2) is used instead ofthe wild type version.

The implications of using a MIFNAR2/ IFNβ non-covalently bound complexaccording to the invention are that lower concentrations of IFNAR2 ECare required and may be used for a variety of therapeutic indications inwhich IFN by itself is therapeutically active.

These indications include those in which free IFNs have shown sometherapeutic activity, such as anti-viral, anti-cancer and immunemodulatory activity. It is expected that the mutant IFNAR2/IFN complex,by virtue of its greater potency, enhanced activity and/or improvedpharmacokinetics (i.e. half-life), will be more efficacious in treatingviral, oncologic and autoimmune disorders.

When administered in vivo the interferon receptor complex enhances thebioavailability, pharmacokinetics, and/or pharmacodynamics of the IFN,thus augmenting the anti-viral, anti-cancer and immune modulatingproperties of the IFN. The preferred molecules for use in the complexesof the present invention comprise the amino acid sequence of native IFNβand MIFNAR2 (SEQ ID NOs: 2, 3, and 4). The native sequence is that of anaturally occurring human IFNβ. Such sequences are known and can bereadily found in the literature. Naturally occurring allelic variationsare also considered to be native sequences.

The present invention also includes analogs of the above MIFNAR2 EC.Such analogs may be ones in which up to about 30, preferably up to 20and most preferably 10 amino acid residues may be deleted, added orsubstituted by others in the proteins, except mutations at residues 78and 100 which results in a decrease in the affinity of MIFNAR2 for IFNβto the wild type IFNAR2 affinity for IFNβ. These analogs are prepared byknown synthesis and/or by site-directed mutagenesis techniques or anyother known technique suitable therefore.

Any such analog preferably has a sequence of amino acids sufficientlyduplicative of that of the basic MIFNAR2 such as to have substantiallysimilar activity thereto. Thus, it can be determined whether any givenanalog has substantially the same activity and/or stability as theprotein and complex of the invention by means of routineexperimentation, comprising subjecting each such analog to binding andbiological activity tests. MIFNAR2 EC analogs may bind IFNβ with atleast 15 fold and about 50 to 100 fold higher affinity over the wildtype protein wherein the affinity towards IFNα2 is not significantlychanged. The MIFNAR2 EC analogs may exhibit a Kd of about 30 pM andlower towards IFNβ. The binding tests for MIFNAR2 and IFN interactionmay involve analytical gel filtration, optical heterogeneous phasedetection (such as surface plasmon resonance [SPR], or reflectometricinterference spectroscopy [RifS] which resembles the widely used BIACOREtechnique) and fluorescent spectroscopy (Piehler and Schreiber 1999APiheler and Schreiber 2001).

Analogs of the complex which can be used in accordance with the presentinvention, or nucleic acid sequence coding therefore, include a finiteset of substantially corresponding sequences as substitution peptides orpolynucleotides which can be routinely obtained by one of ordinary skillin the art, without undue experimentation, based on the teachings andguidance presented herein. For a detailed description of proteinchemistry and structure, see Schulz et al, Principles of ProteinStructure, Springer Verlag, New York (1978); and Creighton, T. E.,Proteins: Structure and Molecular Properties, W.H. Freeman & Co, SanFrancisco (1983), which are hereby incorporated by reference.

For a presentation of nucleotide sequence substitutions, such as codonpreferences, see Ausubel et al (1987, 1992), A.1. I-A. 1.24, andSambrook et al (1987, 1992), 6.3 and 6.4, at Appendices C and D.

Preferred changes for analogs in accordance with the present inventionare what are known as “conservative” substitutions. Conservative aminoacid substitutions of those in the sequence of the proteins in theinvention may include synonymous amino acids within a group, which havesufficient similar physicochemical properties that substitution betweenmembers of the group will preserve the biological function of themolecule (Grantham, 1974). It is clear that insertions and deletions ofamino acids may also be made in the above-defined sequences withoutaltering their function, particularly if the insertions or deletionsonly involve a few amino acids, e.g., under thirty, and preferably underten, and do not remove or displace amino acids which are critical to afunctional conformation, e.g., cysteine residues (Anfinsen, 1973).Analogs produced by such deletions and or insertions come within thepurview of the present invention. Preferably, the synonymous amino acidgroups are those defined in Table I. More preferably, the synonymousamino acid groups are those defined in Table II; and most preferably thesynonymous amino acid groups are those defined in Table III.

TABLE 1 Preferred Groups of Synonymous Amino Acids Amino Acid SynonymousGroup Ser Ser, Thr, Gly, Asn Arg Arg, Gln, Lys, Glu, His Leu Ile, Phe,Tyr, Met, Val, Leu Pro Gly, Ala, Thr, Pro Thr Pro, Ser, Ala, Gly, His,Gln, Thr Ala Gly, Thr, Pro, Ala Val Met, Tyr, Phe, Ile, Leu, Val GlyAla, Thr, Pro, Ser, Gly Ile Met, Tyr, Phe, Val, Leu, Ile Phe Trp, Met,Tyr, Ile, Val, Leu, Phe Tyr Trp, Met, Phe, Ile, Val, Leu, Tyr Cys Ser,Thr, Cys His Glu, Lys, Gln, Thr, Arg, His Gln Glu, Lys, Asn, His, Thr,Arg, Gln Asn Gln, Asp, Ser, Asn Lys Glu, Gln, His, Arg, Lys Asp Glu,Asn, Asp Glu Asp, Lys, Asn, Gln, His, Arg, Glu Met Phe, Ile, Val, Leu,Met Trp Trp

TABLE 2 More Preferred Groups of Synonymous Amino Acids Amino AcidSynonymous Group Ser Ser Arg His, Lys, Arg Leu Leu, Ile, Phe, Met ProAla, Pro Thr Thr Ala Pro, Ala Val Val, Met, Ile Gly Gly Ile Ile, Met,Phe, Val, Leu Phe Met, Tyr, Ile, Leu, Phe Tyr Phe, Tyr Cys Cys, Ser HisHis, Gln, Arg Gln Glu, Gln, His Asn Asp, Asn Lys Lys, Arg Asp Asp, AsnGlu Glu, Gln Met Met, Phe, Ile, Val, Leu Trp Trp

TABLE 3 Most Preferred Groups of Synonymous Amino Acids Amino AcidSynonymous Group Ser Ser Arg Arg Leu Leu, Ile, Met Pro Pro Thr Thr AlaAla Val Val Gly Gly Ile Ile, Met, Leu Phe Phe Tyr Tyr Cys Cys, Ser HisHis Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu Met Met, Ile, Leu Trp Met

Examples of production of amino acid substitutions in proteins which canbe used for obtaining analogs of MIFNAR2 or MIFNAR2 EC for use in thepresent invention include any known method steps, such as presented inU.S. Pat. Nos. RE 33,653; 4,959,314; 4,588,585 and 4,737,462, to Mark etal; U.S. Pat. No. 5,116,943 to Koths et al; U.S. Pat. No. 4,965,195 toNamen et al; and U.S. Pat. No. 5,017,691 to Lee, et al, and lysinesubstituted proteins presented in U.S. Pat. No. 4,904,584 (Shaw et al).

The term “essentially corresponding to” is intended to comprehendanalogs with minor changes to the sequence of the basic MIFNAR2 orMIFNAR2 EC which do not affect the basic characteristics thereof, e.g.,its specific enhanced binding and affinity to IFNβ. The type of changeswhich are generally considered to fall within the “essentiallycorresponding to” language are those which would result fromconventional mutagenesis techniques of the DNA encoding the complex ofthe invention, resulting in a few minor modifications, and screening forthe desired activity in the manner discussed above.

Preferably, the MIFNAR2 portion of the complex will have a core sequencewhich is the same as that of the native sequence or biologically activefragment thereof, or a variant thereof which has an amino acid sequencehaving at least 70% identity to the native amino acid sequence andretains the biological activity thereof More preferably, such a sequencehas at least 85% identity, at least 90% identity, or most preferably atleast 95% identity to the native sequence.

With respect to the IFN portion of the complex, the core sequence whichmay be used is the native sequence, or a biologically active fragmentthereof, or a variant thereof which has an amino acid sequence having atleast 70% identity thereto, more preferably, at least 85% or at least90% identity, and most preferably at least 95% identity. Such analogsmust retain the biological activity of the native IFN sequence orfragment thereof, or have antagonist activity as discussed herein below.

The term “sequence identity” as used herein means that the sequences arecompared as follows. The sequences are aligned using Version 9 of theGenetic Computing Group's GAP (global alignment program), using thedefault (BLOSUM62) matrix (values −4 to +11) with a gap open penalty of−12 (for the first null of a gap) and a gap extension penalty of −4 (pereach additional consecutive null in the gap). After alignment,percentage identity is calculated by expressing the number of matches asa percentage of the number of amino acids in the claimed sequence.

Analogs in accordance with the present invention may also be determinedin accordance with the following procedure. With respect to either theMIFNAR2 portion of the complex or the IFN portion of the complex, theDNA of the IFNAR and IFN sequence are known to the prior art and iseither found in the literature cited in the background section of thepresent specification or can be readily located by those of ordinaryskill in the art. Polypeptides encoded by any nucleic acid, such as DNAor RNA, which hybridizes to the complement of the native DNA or RNAunder highly stringent or moderately stringent conditions, as long asthat polypeptide maintains the biological activity of the nativesequence or, in the case of IFN, either maintains the biologicalactivity of MIFNAR2 or MIFNAR2 EC or possesses antagonistic activity,are also considered to be within the scope of the present invention.

“Stringent conditions” refers to hybridization and subsequent washingconditions, which those of ordinary skill in the art conventionallyrefer to as “stringent”. See Ausubel et al., Current Protocols inMolecular Biology, supra, Interscience, N.Y., §§6.3 and 6.4 (1987,1992), and Sambrook et al. (Sambrook, J. C., Fritsch, E. F., andManiatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.).

Without limitation, examples of stringent conditions include washingconditions 12-20° C. below the calculated Tm of the hybrid under studyin, e.g., 2×SSC and 0.5% SDS for 5 minutes, 2×SSC and 0.1% SDS for 15minutes; 0.1×SSC and 0.5% SDS at 37° C. for 30-60 minutes and then,0.1×SSC and 0.5% SDS at 68° C. for 30-60 minutes. Those of ordinaryskill in this art understand that stringency conditions also depend onthe length of the DNA sequences, oligonucleotide probes (such as 10-40bases) or mixed oligonucleotide probes. If mixed probes are used, it ispreferable to use tetramethyl ammonium chloride (TMAC) instead of SSC.See Ausubel, supra.

“Functional derivatives” as used herein covers derivatives which may beprepared from the functional groups which occur as side chains on theresidues or the N- or C-terminal groups, by means known in the art, andare included in the invention as long as they remain pharmaceuticallyacceptable, i.e., they do not destroy the biological activity of thecorresponding protein of the complex as described herein and do notconfer toxic properties on compositions containing it or the complexmade therefore. Derivatives may have chemical moieties, such ascarbohydrate or phosphate residues, provided such a fraction has thesame biological activity and remains pharmaceutically acceptable.

For example, derivatives may include aliphatic esters of the carboxyl ofthe carboxyl groups, amides of the carboxyl groups by reaction withammonia or with primary or secondary amines, N-acyl derivatives or freeamino groups of the amino acid residues formed with acyl moieties (e.g.,alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of freehydroxyl group (e.g., that of seryl or threonyl residues) formed withacyl moieties. Such derivatives may also include for example,polyethylene glycol side-chains, which may mask antigenic sites andextend the residence of the complex or the portions thereof in bodyfluids.

The term “fused protein” refers to a polypeptide comprising an MIFNAR2,or MIFNAR2 EC or an analog or fragment thereof, fused with anotherprotein, which, e.g., has an extended residence time in body fluids. AnMIFNAR2 or MIFNAR2 EC may thus be fused to another protein, polypeptideor the like, e.g., an immunoglobulin or a fragment thereof.

A “fragment” according to the present invention may, e.g., be a fragmentof MIFNAR2 or MIFNAR2 EC. The term fragment refers to any subset of themolecule, that is, a shorter peptide that retains the desired biologicalactivity. Fragments may readily be prepared by removing amino acids fromeither end of the MIFNAR2 molecule and testing the resultant fragmentfor its properties to bind to IFNβ. Proteases can be used for removingone amino acid at a time from either the N-terminal or the C-terminal ofa polypeptide, and so determining fragments, which retain the desiredbiological activity, involves only routine experimentation.

As active fragments of an MIFNAR2, analogs and fused proteins thereof,the present invention further covers any fragment or precursors of thepolypeptide chain of the protein molecule alone or together withassociated molecules or residues linked thereto, e.g., sugar orphosphate residues, or aggregates of the protein molecule or the sugarresidues by themselves, provided said fragment has substantially similaractivity.

The term “salts” herein refers to both salts of carboxyl groups and toacid addition salts of amino groups of the complex of the invention oranalogs thereof Salts of a carboxyl group may be formed by means knownin the art and include inorganic salts, for example, sodium, calcium,ammonium, ferric or zinc salts, and the like, and salts with organicbases as those formed, for example, with amines, such astriethanolamine, arginine or lysine, piperidine, procaine and the like.Acid addition salts include, for example, salts with mineral acids, suchas, for example, hydrochloric acid or sulfuric acid, and salts withorganic acids, such as, for example, acetic acid or oxalic acid. Ofcourse, any such salts must have substantially similar biologicalactivity to the complex of the invention or its analogs.

The term “biological activity” as used herein is interpreted as follows.Insofar as the MIFNAR2 is concerned, the important biological activityis its ability to bind to IFNβ with increased affinity. Thus, analogs orvariants, salts and functional derivatives must be those chosen so as tomaintain this interferon-binding ability. This can be tested by routinebinding assay experiments. In addition, fragments of the MIFNAR2, oranalogs thereof, can also be used as long as they retain theirinterferon-enhanced binding activity. Fragments may readily be preparedby removing amino acids from either end of the interferon-bindingpolypeptide and testing the resultant for interferon-binding properties.

Additionally, the polypeptide which has such interferon-bindingactivity, be it MIFNAR2, MINFAR2 EC, an analog, functional derivative,or fragment, can also contain additional amino acid residues flankingthe interferon-binding polypeptide. As long as the resultant moleculeretains the increased interferon-binding ability of the corepolypeptide, one can determine whether any such flanking residues affectthe basic and novel characteristics of the core peptide, i.e., itsinterferon-binding characteristics, by routine experimentation. The term“consisting essentially of”, when referring to a specified sequence,means that additional flanking residues can be present which do notaffect the basic and novel characteristic of the specified sequence.This term does not comprehend substitutions, deletions or additionswithin the specified sequence.

While MIFNAR2 or MIFNAR2 EC have been used throughout this descriptionand in the examples, it should be understood that this is merely thepreferred example and that the IFNAR1 subunit, and particularly itsextracellular domain, may be used together with MIFNAR2 or MIFNAR2 EC.

With respect to the interferon part of the complex of the presentinvention, the biological activity which must be maintained in anyanalog, functional derivative, fusion protein or fragment is theactivity of the interferon relied upon for the intended utility. In mostinstances, this will be the ability to bind to a native cell surfacereceptor and thereby mediate signal production by the receptor. Thus,any such analog, derivative or fragment should maintain such receptoragonist activity to be useful in the present invention for such autility. On the other hand, it is sometimes useful to have a moleculewith antagonist activity on the receptor so as to prevent the biologicalactivity of native interferon. Such an antagonist can also be used forprolonged beneficial effect by means of the complex of the presentinvention. For such utilities in which it is desired to eliminate anundesired effect of interferon, analogs which are still bound by thereceptor and by the IFNAR portion of the complex but which do notmediate a signal and block signal generation by the native interferon onthat receptor (i.e., interferon antagonist), may also be considered tobe biologically active for the purpose of this invention and to beencompassed by the term interferon when used with respect to thecomplexes of the present invention. Straightforward assays can determinewhether any such analog maintains such receptor agonist activity or hasreceptor antagonist activity and would, thus, be useful for one of theutilities of the present invention.

The present invention also relates to DNA sequences encoding MIFNAR2 EC,e.g., DNA encoding the amino acid sequences in SEQ ID NOs: 2, 3 and 4 oranalogs, and fragments thereof, as well as DNA vectors carrying such DNAsequences for expression in suitable prokaryotic or eukaryotic hostcells.

The ability to generate large quantities of heterologous proteins usinga recombinant protein expression system has led to the development ofvarious therapeutic agents, e.g., t-PA and EPO (Edington, 1995). Thevarious expression hosts from which recombinant proteins can begenerated range from prokaryotic in origin (e.g., bacteria) (Olins,1993), through lower eukaryotes (e.g., yeast) (Ratner, 1989) to highereukaryotic species (e.g., insect and mammalian cells) (Reuveny, 1993;Reff, 1993). All of these systems rely upon the sameprinciple—introducing the DNA sequence of the protein of interest intothe chosen cell type (in a transient or stable fashion, as an integratedor episomal element) and using the host transcription, translation andtransportation machinery to over-express the introduced DNA sequence asa heterologous protein (Keown, 1990).

Various protocols for the production of recombinant heterologousproteins are described (Ausubel et al., Current Protocols in MolecularBiology, Greene Publications and Wiley Interscience, New York, N.Y.,1987-1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).

In addition to the expression of native gene sequences, the ability tomanipulate DNA at the nucleotide level has expedited the development ofnovel engineered sequences which, although based on natural proteins,possess novel activities as a result of the alteration in primaryprotein structure (Grazia, 1997).

Moreover, chosen sequences of DNA can be physically linked to generatetranscripts which develop into novel fusion proteins where onceindependent proteins are now expressed as one polypeptide unit (Ibanez,1991). The activity of such fusion proteins can be different, e.g., morepotent, than either of the individual proteins (Curtis, 1991).

For co-administration of MIFNAR2 EC with IFN, human IFNβ may be derivedfrom a production process, which uses the mammalian Chinese hamsterovary cell (CHO) as disclosed in EP220574. Type 1 interferons can beexpressed in a variety of host cells including those of bacteria(Utsumi, 1987), insect (Smith, 1983) and human (Christofinis, 1981)origin. Also human MIFNAR2 or a fragment thereof may be expressed usingthe CHO host cell. For secretion of MIFNAR2 EC from CHO cells, MIFNAR2EC DNA sequence may be ligated to the sequence of the human growthhormone signal peptide as described in the patent application WO0022146.Alternatively, soluble receptors, such as MIFNAR2 EC, may be expressedsuccessfully in bacterial expression systems (Terlizzese, 1996).

The invention also relates to a pharmaceutical composition comprising asactive ingredient an MIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFN complex oranalogs, fusion proteins, functional derivatives, fragments thereof ormixtures thereof or salts thereof and a pharmaceutical acceptablecarrier, diluent or excipient. An embodiment of the pharmaceuticalcomposition of the invention includes a pharmaceutical composition forenhanced IFN type action, in the treatment of viral diseases, inanti-cancer therapy, in immune modulation therapy, e.g., in autoimmunediseases and other applications of interferons and cytokines relatedthereto.

The pharmaceutical compositions of the invention are prepared foradministration by mixing an MIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFN complexor analogs, fusion proteins, functional derivatives, fragments thereofor mixtures thereof or salts thereof with physiologically acceptablestabilizers and/or excipients, and prepared in dosage form, e.g., bylyophilization in dosage vials. The method of administration can be viaany of the accepted modes of administration for similar agents and willdepend on the condition to be treated, e.g., intravenously,intramuscularly, and subcutaneously, by local injection or topicalapplication, or continuously by infusion, etc. The amount of activecompound to be administered will depend on the route of administration,the disease to be treated and the condition of the patient.

The invention relates to a method for treatment of autoimmune diseasessuch as multiple sclerosis, rheumatoid arthritis, myasthenia gravis,diabetes, lupus and ulcerative colitis, comprising administration of atherapeutically effective amount of an MIFNAR2, MIFNAR2 EC, MIFNAR2EC/IFN complex or analogs, fusion proteins, functional derivatives,fragments thereof or mixtures thereof or salts thereof.

The invention relates to a method for treatment of a viral disease suchas granulomatous disease, condyloma acuminatum, juvenile laryngealpapillomatosis, hepatitis A or chronic infection with hepatitis B and Cviruses, comprising administration of a therapeutically effective amountof an MIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFN complex or analogs, fusionproteins, functional derivatives, fragments thereof or mixtures thereofor salts thereof.

The invention relates to a method for treatment of various types ofcancer such as hairy cell leukemia, Kaposi's sarcoma, multiple myeloma,chronic myelogenous leukemia, non-Hodgkins's lymphoma or melanoma,comprising administration of a therapeutically effective amount of anMIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFN complex or analogs, fusion proteins,functional derivatives, fragments thereof or mixtures thereof or saltsthereof.

In the above methods an MIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFN complex oranalogs, fusion proteins, functional derivatives, fragments thereof ormixtures thereof or salts thereof may be administered together with IFN,preferably IFNβ.

A “therapeutically effective amount” is such that when administered, anMIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFN complex or analogs, fusion proteins,functional derivatives, fragments thereof or mixtures thereof or saltsthereof results in modulation of the biological activity of IFNβ. Thedosage administered, as single or multiple doses, to an individual mayvary depending upon a variety of factors, including the route ofadministration, patient conditions and characteristics (sex, age, bodyweight, health, size), extent of symptoms, concurrent treatments,frequency of treatment and the effect desired. Adjustment andmanipulation of established dosage ranges are well within the ability ofthose skilled in the art, as well as in vitro and in vivo methods ofdetermining the activity of an MIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFNcomplex or analogs, fusion proteins, functional derivatives, fragmentsthereof or mixtures thereof or salts thereof.

Local injection, for instance, will require a lower amount of theprotein on a body weight basis than will intravenous infusion.

Free IFNβ has a tendency to oligomerize. To suppress this tendency,present day formulations of IFNβ have an acidic pH, which may cause somelocalized irritation when administered. As an MIFNAR2, MIFNAR2 EC, oranalogs, fusion proteins, functional derivatives, fragments thereof ormixtures thereof or salts thereof can serve as a superior stabilizerover the wild type version for IFNβ and thereby prevent oligomerization,its use in IFNβ formulations can serve to stabilize the IFNβ and therebyobviate the necessity of acidic formulations. Accordingly, a non-acidicpharmaceutical composition containing an MIFNAR2, MIFNAR2 EC, oranalogs, fusion proteins, functional derivatives, fragments thereof ormixtures thereof or salts thereof, along with other conventionalpharmaceutically acceptable excipients, is also a part of the presentinvention.

The present invention also includes uses of an MIFNAR2, MIFNAR2 EC,MIFNAR2 EC/IFN complex or analogs, fusion proteins, functionalderivatives, fragments thereof or mixtures thereof or salts thereof foranti-viral, anti-cancer and immune modulation therapy. Specifically, themutant interferon receptor and interferon complexes of this inventionare useful for anti-viral therapy in such therapeutic indications aschronic granulomatous disease, condyloma acuminatum, juvenile laryngealpapillomatosis, hepatitis A and chronic infection with hepatitis B and Cviruses.

In particular, the mutant interferon receptor and interferon complexesof this invention are useful for anti-cancer therapy in such therapeuticindications as hairy cell leukemia, Kaposi's sarcoma, multiple myeloma,chronic myelogenous leukemia, non-Hodgkins's lymphoma and melanoma.

The mutant interferon receptor and interferon complexes of thisinvention are also useful for immune modulation therapy, in autoimmunediseases, e.g., multiple sclerosis, rheumatoid arthritis, myastheniagravis, diabetes, lupus, ulcerative colitis etc.

“An autoimmune disorder” is a disease in which a person's immune systembegins to attack his or her own body. The immune system createsantibodies against its own tissues. Virtually every part of the body issusceptible to an autoimmune disorder.

The mutant interferon receptor and interferon complexes are also usefulfor treating neurodegenerative diseases, preferably multiple sclerosis.

The invention further relates to a pharmaceutical composition comprisingan MIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFN complex or analogs, fusionproteins, functional derivatives, fragments thereof or mixtures thereofor salts thereof, to a pharmaceutical composition comprising anexpression vector, in particular a lentiviral gene therapy vectorexpressing an, MIFNAR2, MIFNAR2 EC, MIFNAR2 EC/IFN complex or analogs,fusion proteins, fragments thereof.

The terms “treating” as used herein should be understood as preventing,inhibiting, attenuating, ameliorating or reversing any or all symptomsor cause(s) of the disease.

Having now described the invention, it will be more readily understoodby reference to the following examples that are provided by way ofillustration and are not intended to be limiting of the presentinvention.

EXAMPLES Example 1 Protein Expression and Purification

IFNAR2-EC (extracellular domain) and IFNα was expressed in E. colipurified by ion exchange and size-exclusion chromatography as described(Piehler & Schreiber, 1999A). The levels of expression of IFNAR2-ECmutants were as high as the wild type. Wild type, glycosylated IFNβ wasproduced in CHO (disclosed in EP220574). Protein concentrations weredetermined from absorbance at 280 nm (Piehler & Schreiber, 1999A) with1:280=18,070 M-1 for IFNα2, 1:280=30,050 M-1 for 1FNβ and 1:280=26,500M-1 for IFNAR2-EC (corrected to 1:280=21,100 M-1 for the tryptophanmutants of IFNAR2-EC W102A and W74F). Protein purity was analyzed bySDS-PAGE under non-reducing conditions.

Example 2 Generation of IFNAR EC Mutants

Site-directed mutagenesis was carried out by PCR with the templatepT72CR2 (Piehler and Schreiber 1999) and with 18-21 nucleotide primerscontaining the mutated codon using high fidelity polymerases pwo(Boehringer Mannheim) and Pfu (Stratagene) as described in detail(Albeck & Schreiber, 1999). After phosphorylation and ligation, themutated plasmids were used to transform E. coli TG1 cells. The sequenceof the whole expressed gene containing the mutation was verified by DNAsequencing (Ausubel et al., Current Protocols in Molecular Biology,Greene Publications and Wiley Interscience, New York, N.Y., 1987-1995;Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989).

Mutants were generated in which two amino acid residues, histidine 78(H78) and Asparagine 100 (N100), were mutated: A—both to alanineresidues (H78A/N100A mutant), B—to alanine and aspartic acidrespectively (H78A/N100D) and C—to alanine and histidine respectively(H78A/N100H).

Example 3 Thermodynamic and Kinetic Analysis

All thermodynamic and kinetic data were obtained from label-freeheterogeneous phase detection. The interaction between IFNβ andIFNAR2-EC was monitored by reflectometric interference spectroscopy(RifS) under flow-through conditions as described (Piehler & Schreiber,1999A). This technique is similar to Biacore and is used to accuratelymeasure affinity of binding between two proteins. IFNAR2-EC (wild-typeor mutant) was immobilized trough immobilized specific antibodies (asdescribed by Piehler and Schreiber 2001). All measurements with IFNβ,IFNα2 and IFNAR2-EC were carried out in 50 mM Hepes with 500 mM NaCI and0.01% Triton X100 at pH 7.4. The interaction was measured at 500 mM NaClin order to eliminate non-specific interactions with the surface, whichwas observed with IFNβ at 150 mM NaCI.

Association and dissociation kinetics were measured by standardinjection protocols and corrected by blank runs. Dissociation rateconstants were measured at IFN concentration in the range of 1-1000 nMin order to saturate the surface. The total range of dissociation wasused for fitting a 1:1 kinetic model (Piehler & Schreiber, 2001).

Example 4 Anti-Viral Activity Assay

Anti-viral activity of IFNβ was assayed as the inhibition of thecytophatic effect of vesicular stomatitis virus (VSV) on human WISHcells (Rubinstein et al., 1981).

Example 5 Measurement of IFN Binding to Mutant IFNAR2

Binding of the IFNβ and IFNα2 to the H78A/N100A mutant (example 2) wasmeasured and compared to the wild type EC receptor by RifS (example 3).While the association rate of IFNβ to the H78A/N100A mutant was found tobe similar to that of the wild type (FIG. 3) the disassociation rate wasfound to be significantly lower. The calculated affinity of IFNβ toH78A/N100A mutant is about 30 pM versus the affinity to the WT proteinof about 3 nM. In contrast to IFNβ, both the association anddisassociation rate of IFNα2 to the H78A/N100A mutant, were found to besimilar to the rates obtained with the wild type protein (FIG. 3). Theseresults show that the affinity of the IFNAR2 mutant was found to beapproximately 100 times higher than the wild type towards IFNβ andunchanged towards IFNα2.

Example 6 Relative Affinities of Interferon Towards the Mutant IFNAR2

The binding and affinities of IFNAR EC receptor and mutant receptor EC(example 2) to IFNβ and IFNα2 were measured using RifS, with IFNAR2 wildtype or mutant immobilized to the surface trough specific antibodies(example 3). After measuring the affinities, the relative affinitieswere obtained by comparing the Kd of the mutant receptor over the Kd ofthe wild type receptor (Table 4).

The Kd of binding of interferon to IFNAR2 extracellular domain (EC) wasmeasured by RifS and was found to be about 3nM (example 5). The Kd ofIFNβ binding to H78A/N100A (EC) mutant was about 30 pM. The exactmeasurement of Kd for this mutant was not possible, because binding wasto tight to get good data from RifS. The Kd of IFNα2 to the H78A/N100AEC mutant was found to be similar to the wild type receptor. The resultsin Table 4 show the relative affinities of the IFNAR EC mutants comparedto the wild type IFNAR2 receptor EC. The mutants were the following:mutated in one amino acid residue, H78A or N100A, and mutated in twoamino acids H78A/N100A, H78A/N100D and H78A/N100H, wherein the aminoacid N100 is mutated into alanine, aspartic acid or histidinerespectively (example 2). The results demonstrate that the singlemutations in IFNAR2 increase the affinity of the complex from 4.6 up to7.3 fold, while the double mutation causes a synergistic effect,increasing the affinity of the complex by 26 and to above 50-fold. Thebest mutant in terms of affinity was found to be the double mutant withthe N100 modified to alanine, exhibiting over 50 fold increased affinityversus the wild type version.

TABLE 4 IFNAR2 IFNα2 IFNβ wt 1.0 1.0 H78A 0.4 4.6 N100A 2.0 7.3H78A/N100A 0.7 >50 H78A/N100D 1.0 40.0 H78A/N100H 0.9 26.0

Example 7 Occlusion of Interferon Beta by the IFNAR2 Mutant

The capability of IFNAR2 EC wild type and mutants EC to serve ascarriers of IFNβ was compared. For that purpose antiviral activity ofIFNβ residual (free) in samples comprising a constant concentration ofIFNβ (10 pM) mixed with varying concentrations of recombinant solubleIFNAR2 EC or IFNAR2 mutants EC (example 6) was monitored. In theantiviral assay, the mixture (IFNAR2/IFN complex) was added to WISHcells (human amniotic cells). These WISH cells were then challenged withvesicular stomatitis virus (VSV), and the residual (free) anti-viralactivity of IFNβ was monitored as the degree of cell survival following24-hour incubation (example 4). The free IFNβ present in samples havingdifferent amount of WT or mutant IFNAR2 EC (R2) concentration wasdetermined from a survival dose curve of antiviral activity as afunction of IFNβ concentration carried out in the absence of IFNAR2(FIG. 4 upper plot).

The mutants tested were the following: IFNAR2 EC mutated in one aminoacid residue, H78A or N100A, and mutated in two amino acids H78A/N100A,H78A/N100D and H78A/N100H wherein the amino acid N100 is mutated intoalanine, aspartic acid and histidine respectively (example 2). Thedouble mutant of IFNAR2 H78A/N100A (example 2) showed the highestaffinity of all the generated mutants (Kd of about 30 pM and lower, seeexamples 5 and 6).

FIG. 4 shows that in the presence of 2.5 nM of wild type IFNAR2 EC about20% IFNβ is bound to the soluble receptor (occluded), while in thepresence of only 0.2 nM of the double mutant EC H78A/N100A 50% of IFNβis bound and using only 0.4 nM of H78A/N100A mutant EC 80% of the IFNβis bound. The biological assay demonstrated also, that the same extentof occluded IFNβ (bound IFNβ under equilibrium conditions) and theresidual antiviral activity (free IFNβ) obtainable with wild type IFNAR2could be accomplished using about 30 fold lower concentration of theH78A/N100A IFNAR2 mutant EC. The results show also that the doublemodified mutants yield the best results, particularly the one in whichboth amino acids were mutated to alanine, H78A/N100A IFNAR2.

This result shows that the double mutated IFNAR2 occlude moreeffectively IFNβ and therefore administration of considerably loweramounts will be required to accomplish its carrier activity.

REFERENCES

Alam et al, Pharmaceutical Research 14:546-549 (1997).

Anfinsen, Science 181:223-230 (1973).

Ausubel et al, Current Protocols in Molecular Biology, GreenePublications and Wiley Interscience (New York, 1987-1992).

Baron et al, Antiviral Res. 24:97-110 (1994).

Baron, et al, .J. Am. Med. Assoc. 266:1375-1383 (1991).

Christofinis, G. J Journal of General Virology 52:169-171 (1981).

Colamonici et al, J. Immunol. 148:2126-2132 (1992).

Colamonici et al, J. Biol. Chem. 268:10895-10899 (1993).

Curtis, B. M., Proc. Natl. Acad. Sci. 88:5809-5813 (1991).

Domanski et al, The Journal of Biological Chemistry 270:6 (1995).

Duncan et al, J. Exp. Med. 184:2043-2048 (1996).

Dron et al, “Interferon a/f3 gene structure and regulation” inInterferon: Principles and Medical Applications, Baron et al, Editors,(University of Texas Medical Branch: Galveston, Tex., 1992) pp. 33-45

Edington, S. M., “Biotech Products as Drug Leads” BioTechnology 13:649(1995).

Fierlbeck et al, Journal of Interferon and Cytokine Research 16:777(1996).

Grantham, Science 185:X62-X64 (1974).

Grazia Cusi, Mo, Immunotechnoloqy 3:61-69 (1997).

Ibanez, C. F., EMBO Journal 10:2105-2110 (1991).

Jacobs L, et al. Arch Neurol 1987 Jun; 44(6):589-95.

Keown, W. A., Methods in Enzymology 185:527-537 (1990).

Lengyl, P. Ann. Rev. Biochem. 51:251-282 (1982).

Lutfalla et al, EMBO Journal 14:5100-5108 (1995).

Novick et al, FEBS Lett. 314:445-448 (1992).

Novick et al, Cell 77:391-400 (1994).

Novick et al, J. Leuk. Bio. 57:712-718 (1995).

Piehler and Schreiber J. Mol. Biol. 1999A 289, 57-67.

Piehler and Schreiber J. Mol. Biol. 1999B 294, 223-237.

Piehler and Schreiber Analytical Biochemistry 289, 173-186.

Platanias et al, 1993 J. Immunology 150 : 3382-3388.

Platanias et al, 1996 J. Biol. Chem. 271 : 23630-3.

Salmon et al, 1996 Journal of Interferon and Cytokine Research 16 : 759.

Sharf et al, 1995 J. Biol. Chem. 270: 13063-9.

Tan et al, 1973. J. Exp. Med. 137: 317-330.

Uze et al, 1990 Cell 60 : 225-34.

Yang et al, 1996 J. Biol. Chem. 271 : 8057-61.

Yan et al., 1996 Mol. Cell Bio. 16 : 2074-82.

1. A method for augmenting the anti-cancer, immune modulating oranti-viral properties of interferon-β (IFNβ) comprising administering toa patient in need thereof a therapeutically effective amount of acomposition comprising a fusion protein comprising (1) an IFNAR2 portionconsisting of the sequence of SEQ ID NO: 2 and (2) an immunoglobulinportion consisting of an immunoglobulin or fragment thereof, wherein theaffinity of said fusion protein for IFN-β is synergistically increased25 to 100-fold compared to the affinity of wild type human IFNAR2 forIFN-β.
 2. The method of claim 1, wherein the composition furthercomprises IFNβ.
 3. The method of claim 1 or 2, wherein the method is forthe treatment of an autoimmune disease selected from multiple sclerosis,rheumatoid arthritis, myasthenia gravis, diabetes, lupus and ulcerativecolitis.
 4. The method of claim 1 or 2, wherein the method is for thetreatment of a cancer selected from hairy cell leukemia, Kaposi'ssarcoma, multiple myeloma, chronic myelogenous leukemia, non-Hodgkin'slymphoma and melanoma.
 5. The method of claim 1 or 2, wherein the methodis for the treatment of a viral disease selected from the groupconsisting of chronic granulomatous disease, condyloma acuminatum,juvenile laryngeal papillomatosis, hepatitis A, and chronic infectionwith hepatitis B and C viruses.
 6. The method of claim 2, wherein thefusion protein and IFNβ are covalently linked.
 7. A method foraugmenting the anti-cancer, immune modulating or anti-viral propertiesof IFNβ comprising administering to a patient in need thereof atherapeutically effective amount of a composition comprising an isolatedpolypeptide consisting of the sequence of SEQ ID NO:
 2. 8. The method ofclaim 7, wherein the composition further comprises IFNβ.
 9. The methodof claim 7 or 8, wherein the method is for the treatment of anautoimmune disease selected from multiple sclerosis, rheumatoidarthritis, myasthenia gravis, diabetes, lupus and ulcerative colitis.10. The method of claim 7 or 8, wherein the method is for the treatmentof a cancer selected from hairy cell leukemia, Kaposi's sarcoma,multiple myeloma, chronic myelogenous leukemia, non-Hodgkin's lymphomaand melanoma.
 11. The method of claim 7 or 8, wherein the method is forthe treatment of a viral disease selected from the group consisting ofchronic granulomatous disease, condyloma acuminatum, juvenile laryngealpapillomatosis, hepatitis A, and chronic infection with hepatitis B andC viruses.
 12. The method of claim 8, wherein the polypeptide and IFNβare covalently linked.
 13. A method for inhibiting the activity of IFNβcomprising administering to a patient in need thereof a therapeuticallyeffective amount of a composition comprising a fusion protein comprising(1) an IFNAR2 portion consisting of the sequence of SEQ ID NO: 2 and (2)an immunoglobulin portion consisting of an immunoglobulin or fragmentthereof, wherein the affinity of said fusion protein for IFN-β issynergistically increased 25 to 100-fold compared to the affinity ofwild type human IFNAR2 for IFN-β.
 14. A method for inhibiting theactivity of IFNβ comprising administering to a patient in need thereof atherapeutically effective amount of a composition comprising an isolatedpolypeptide consisting of the sequence of SEQ ID NO:
 2. 15. The methodof claim 13 or 14 wherein the composition further comprises an IFNβantagonist.